Amino acid amides of pteroic acid and processes of preparing the same



Patented Feb. 6, 1951 AMINO ACID AMIDES OF PTEROIC ACID AND PROCESSES OFPREPARING THE SAME James F. McPherson, Elizabeth, N. 3., and RalphMozingo, Minneapolis, Minn assignors to Merck & (30., inc., Bahway, N.3., a corporation of New Jersey No Drawing. Application September 28,1948, Serial No. 51,644

This invention relates to chemical compounds having growth promotingaction for certain organisms and to processes for preparing suchcompounds.

More particularly, the invention relates to procedures by whichsubstituted amides of p-[ (Z-amino 4 hydroxypyrimido[4,5-blpyrazine-S-ylmethyl)amino]-benzoic acid can be preparedsynthetically. Of the various amides that may be obtained, the mostimportant appear to be those of the a-amino acids, and particularly ofglutamic acid.

The glutamic acid amide, N-(p-[(2-amino-4- hydroxypyrimido [4,5-b]-pyrazine-6-y1methyl) aminol-benzoyD-glutamic acid, is a pterin-likeacidic chemical compound which is essential for the growth ofLactobacillus casei and also possesses hematopoietic activity foranimals. Other a-amino acid amides also have growth promoting action andpossess other important properties.

In a co-pending application of one of us, Mozingo, and our colleagues,Serial No. 739,248, filed April 3, 1947, processes for the preparationof N- (p- (2-amino-4-hydroxypyrimido [4,5-b] pyrazine-G-ylmethyl)-amino] -benzoyl) -glutamic acid, referred to therein asN-[4-(UZ-amino-4- hydroxy- B-pteridyl) -methyl] amino) benzoyl] glutamicacid, are disclosed wherein 4-([(2-acyl-' amino 4 hydroxy- 6 -pteridyl)methyl] -acy1- amino benzoie acid, referred to herein as p-[(2-acylamino-4-hydroxypyrimido [4,5-bl-pyrazine- G-ylmethyl)-acylamino]-benzoic acid, is con verted to the corresponding benzoylhalide,

9 Claims. (Cl. 260251.5) Y

reacted with a glutamic acid ester in the presence of an alkalinecondensing agent such as an alkali metal hydroxide, pyridine, ormixtures of both, and hydrolyzed to yield the desired compound.

it has now been discovered in accordance with the present invention thatthe above-reaction between a benzoyl halide and a glutamic acid ester,or other u-amino acid ester, can be carried out Without the aid of analkaline condensing agent, and that when the reaction takes place withboth compounds in solution in a partially halogenated hydrocarbon, asubstantial increase in yield of pure N (p-[(2amin0-l-hydroxypyrimido[4,5-b] -pyrazine-6 ylmethyl) -aminol benzoyl)a-an1ino acid is obtained.In the case of the glutamic acid derivative, the increase in yield ofpure product amounts to about 20-30%, and. a pure product is more easilyobtainable.

Accordingly, the process of this invention involves convertingp-l(2-acylaminoi-hydroxypyrimido [4,5 b] pyrazine-G-ylmethyl)-acylamino] -benzoic acid to the corresponding benzoyl ha-ide, reactingsaid benzoyl halide with an a.- amino acid ester while both compoundsare in solution in a partially halogenated hydrocarbon, hydrolyzing theresulting compound, and recovering an N- (p- (2-amino-4-hydroxypyrimido-[4,5-b] -pyrazine- 6 ylmethyl) -amino] -benzoyl) a-amino acid. Thereactions may be chemically represented, employing a glutamic acid esteras one of the starting materials, as follows:

Wherein X represents a halogen, R represents an acyl group containingtwo or more carbon atoms, and R represents an alkyl or aralkyl group.

In this process, we utilize as one of the starting materials p-[(2-acylamino-l-hydroxypyrimido [4,5-b] pyrazine-G-ylmethyl)acylaminolbenzoic acid (I). This compound may be obtained by acylatingp-[(2-amino-4-hydroxy-pyrimido [4,5-bl-pyrazine 6 ylmethyl) amino]-benzoic acid in accordance with the precedure described in a co-pendingapplication of our colleagues, Wolf and Folkers, Serial No. 689,814,filed August 10, 1946, now Patent No. 2,515,813, We have found that theacyl group employed should contain two or more carbon atoms. Examples ofacyl groups that can be used are acetyl, propionyl, butyryl, isobutyryl,valeryl, benzoyl, phenylacetyl, and the like.

The other starting material, namely an ester of an a-amino acid, may beeither an alkyl or an aralkyl ester, and any one of the d, l, and dlforms may be employed. The amino acid may be any of the a-amino acids,such as arginine, lysine, proline, methionine, leucine, phenylalanine,valine, aspartic acid, alanine, and the like, including polypeptidessuch as glutamylglutamic acid, glutamylglutamylglutamic acid,glycylalanine, and the like.

In carrying out the process of the present invention, compound I isreacted with a thionyl halide by heating them together. Removal of theexcess thionyl halide by evaporation leaves a residue containing p- E(z-acylamino-e-hydroxypyrimido [4,543] -pyrazine-6-ylmethyl) -acylaminol-benzoyl halide (1D. This material may be used directly in thefollowin reaction or may be further purified by repeated precipitationfrom chloroform solution with petroleum ether. We prefer to purifycompound II in this manner before proceeding, as generally higher yieldsof purer product are then obtained.

We have found that the amino acid ester is most suitably reacted withcompound II when both compounds are in solution in a partiallyhalogenated hydrocarbon or mixtures of such hydrocarbons selected fromthe group consisting of chlorinated and brominated lower alkanes andlower alkenes containing one of the radicals wherein X represents ahalogen selected from the group consisting of chlorine and bromine. Thehalogenated hydrocarbon employed should be a liquid at room temperature.Examples of suitable solvents are chloroform, bromoform,dichloromethane, dibromomethane, dichloroethane, dibromoethane,trichloroethane (1,1,2) trichloroethylene, trichlorodibromoethane(1,1,l;2,2), dibromopropane (1, dibromopropane (1,2), tribromobutane(1,2,3), and the like.

The amino acid ester is ordinarily obtained from its mineral acid saltin one of several ways. One method involves dissolving the acid salt inwater, adding sodium bicarbonate to the solution, and extracting withthe reaction solvent to be employed. An alternative method involvesdissolvin the acid salt in the reaction solvent containing thetheoretical amount of pyridine required to neutralize the acid. Thislatter method is not preferred, inasmuch as the presence of pyridine inthe following reaction mixture results in slightly lower yields.

The solution of amino acid ester dissolved in a halogenated hydrocarbonis added to the solid compound II to eifect a reaction whereby an N- (pi(2-acylamino-4-hydroxypyrimido [4,5-bl pyrazine-6-ylmethyl) acylaminol-benzoyl) -amino acid ester (corresponding to compound III) is formed.The amino acid ester removes the hydrochloric acid formed in thereaction, and for that reason and to prevent further acylation of theamino acid ester, at least two molar equivalents of this ester areordinarily employed with one molar equivalent of compound II. We preferto use about three or four molar equivalents of ester per molarequivalent of compound II, for we find that the most favorable yieldsand quality are then obtained. The required amount of solvent is thatnecessary to efiect solution of the reactants, and additional solventcan be used if desired. The reaction will take place from about 0 C. toabout C., or the boiling point of the solution when lower. We prefer tooperate at about room temperature due to the simplicity of operation.Most of the reaction takes place in a relatively short time, but weprefer to allow the reaction mixture to stand about 12-24 hours toachieve more cornplete reaction. When the standing period is complete,the solvent is removed by evaporation, resulting in a residue containinga compound such as compound III.

This compound is then hydrolyzed by dissolving and heating in an aqueousmedium containing slightly more than the theoretical amount of alkalimetal hydroxide necessary to effect the desired hydrolysis, resulting inan N-(p-[(2- amino l hydroxypyrimido [d,5-bl-pyrazine-6- ylmethyl)amino] benzoyl) amino acid (corresponding to compound IV). The reactionis preferably carried out at about the boiling point of the solution toefiect the most rapid hydrolysis, though lower temperatures can beemployed if desired. The hydrolysis period should be only that necessaryto complete the desired hydrolysis, the preferential hydrolysis of thetwo original acylamino groups to amino groups and of the ester group orgroups to an acid group or groups, for increasing this period may resultin further hydrolysis or cleavage. When a compound corresponding tocompound II is hydrolyzed with aqueous sodium hydroxide, the optimumhydrolysis period is about 3 hours at reflux temperature.

Acidification of the resulting solution to a pH of about 2-4 (i. e. toabout the isoelectric point of the compound) results in theprecipitation of the crude compound such as compound IV. Where furtherpurification is desired, the precipitate is dissolved in a mineral acidand crystallized by adjusting the pH of the solution to 3-5. Thisprocedure is repeated where higher purity is desired. We prefer to usean alkali metal salt or" a weak acid, such as sodium acetate, to adjustthe pH for the reason that bet er crystallization results than when abase such as an alkali metal hydroxide is used. Still higher purity andbest crystals of the product are obtainable by recrystallizing fromaqueous solution the material previously precipitated from acidsolution.

The following examples illustrate methods of carrying out the presentinvention, but it is t be understood that these examples are givenprimarily by way of illustration and not of limitation.

Example 1 A mixture of 1 gram (0.0025 mole) of p-E(2- assa-274Zine-S-ylmethyl)-acetamidol-benzoic acid and 25 milliliters of thionylchloride was warmed on the steam bath for five minutes until a clearyellow-orange solution formed. The solvent was removed under reducedpressure to dryness yielding p-[ (2-acetainido-4-hydroxypyrimido [4,5-blpyrazine-(i-ylmethyl) -acetamido] -benzoyl chloride as a yellow-orangepowder.

A chloroform solution of l(+)glutamic acid diethyl ester was thenprepared: 1.72 grams (0.0072 mole) of l(+)glutamic acid diethyl esterhydrochloride was dissolved in 40 milliliters of water, 0.6 gram (0.0072mole) of sodium bicarbonate was added, and the solution was extractedwith four 40-milliliter portions of chloroform. The chloroform extractswere dried over anhydrous magnesium sulfate, filtered and concentratedunder reduced pressure to a 30-milliliter volume.

The chloroform solution of l(+)glutamic acid diethyl ester was added tothe solid p-[(2-acetamido 4 hydroxypyrimido [4,5 b] pyrazine-6-y1methyl)-acetamidol-benzoyl chloride with stirring. Suificientchloroform was then added to the mixture to make the final volume about50 milliliters. A yellow-orange solution developed at once. Stirring wascontinued for several hours and the solution was allowed to stand forfour days at room temperature. The solvent was removed under reducedpressure leaving a fluffy solid containingN-(p-[(2-acetamido-4-hydroxypyrimido [4,5 b] pyrazine 6 ylmethyl)-acetamidol-benzoyl) -glutamic acid diethyl ester.

This fiuify solid was dissolved in 50milliliters of N-so-dium hydroxideand further evacuated to remove last traces of chloroform. The solutionwas heated at a bath temperature of 120 C. for three hours, cooled anddiluted to 100-milliliter volume. After acidification to pH 2-3 withconcentrated hydrochloric acid, the precipitate which formed wascollected by centrifugation and was washed successively with watercontaining a little acetic acid, acetone and ether. A yellow-tan produotwas secured that weighed 0.94 gram and was about 60%N-(p-[(2-amino-4-hydroxypyrimido [4,5 b] pyrazine 6- ylmethyl) amino]-benzoyl) -glutamic acid, a yield of about 51% of theory.

Nine-tenths of a gram of the 0.94 gram obtained above was extracted withtwo 20-milliliter portions of hydrochloric acid. A residue was leftwhich weighed 0.25 grams. Dilution with water to a 400-milliliter volumeof the 40-milliliter acid extract deposited a small amount of solidwhich was removed. To the yellow 400-milliliter volume was added sodiumacetate to pH 3-4 until no more yellow-orange precipitate formed. Thisprecipitate was collected by centrifugation and was washed with watercontaining a little acetic acid, and then with acetone and finallyether. There was secured 0.5 gram of a yelloworange product which wasessentially pure N- (p [(2 amino 4 hydroxy pyrimido [4,5- bl-pyrazine 6ylmethyl) amino] benzoyl) glutamic acid as shown by comparison bioassayswith natural N- (p- (2-amino-4-hydroxypyrimido [4,5 b] pyrazine6-ylmethyl) amino] benzoyl) -glutamic acid, representing a yield ofabout 47% of theory.

Example 2 methyl) -acetamido] -benzoic acid, yellow-orange.

' "p-[ (2- acetamido 4- hydroxypyrimido [4 540]- 4-hydroxypyrimidopyrazine-6yimethyl)'-acetamidol -benzoyl chloride was prepared as inExample 1.

A chloroform solution of l(+) glutamic'acid diethyl ester was preparedby dissolving "5.7 grams (0.024 mole) of l(+)glutamic acid diethyl esterhydrochloride in 50 milliliters of water, adding 2.0 grams (0.024 mole)of sodium bicarbonate to the'solution and extracting with four60-milliliter portions of chloroform. The chloroform'solution was driedover anhydrous magnesium sulfate, filtered and concentrated underreducedpressure to a 50-milliliter volume.

The chloroform 'solutioncontaining l(+)glutamic acid diethyl ester wasadded to the solid p-[ (2-acetamido-4-hydroxypyrimido [4,5-b]-pyrazine-c-ylmethyl) acetamidol-benzoyl chloride with stirring and ayellow-orange solution immediately formed. After stirring the mixturefor 20 hours, the solvent was removed in vacuo almost completely toleave a reddish gum that weighed about 6.0 grams and contained N-(p- [(2acetamido-4hydroxypyrimido [4,5-b] -py1'- azine 6 ylmethyl) acetamido]benzoyl) -glutamic acid diethyl ester.

The reddish gum obtained above was dissolved in 50 milliliters of lN-sodium hydroxide and evacuated to remove the last of the chloroform.The solution was heated for three hours at a bath temperature of 120 0.,cooled and diluted to milliliters with water. Acidification of thesolution to pH 2 with concentrated hydrochloric acid resulted in theformation of a precipitate which was collected and washed successivelywith water containing a small amount of acetic acid, acetone and ether.A product was secured that weighed 0.92 gram and was approximately 48%N- (p[ (2-amino-4-hydroxypyrimido [4,5-b] -pyrazine 6 ylmethyl) amino]-benzoyl) -glutamic acid as shown by bioassay comparison with naturalN-(p-[ (2-amino 4 hydroxypyrimido [4,5- bl pyrazine-B-ylmethyl) -arnino]-benzoyl) -glutamic acid, a yield of about 40% of theory.

Purification of the crude product by the extraction procedure in Example1 gave a total of 0.59 gram of about 60%N-(p-[(2-amino-4-hydroxypyrimido [4,5-bl-pyrazine 6 ylmethyl)aminol-benzoyD-glutamic acid, a yield of about 32% of theory. Theultraviolet absorption spectrum of this compound in pH 10.81 solutionshowed characteristic maxima at 3075 .4, E17?" 1cs;-2s25 A, an, 54.0;

25.50 24., Egg- 538 Example 3 From-1 gram (0.0025 mole) of p-[("-acetamido- [4,540] pyrazine-G-yimethyD-acetamidol-benzoic acid,yellow-orange p- [(2-acetamido 4 hydroxypyri'mido [4,5-bl-pyrazine-6-ylmethyl)-acetamidol benzoyl chloride was prepared as inExample 1.

A chloroform solution of l(+) glutamic acid di ethyl ester was preparedby dissolving 1.72 grams (0.0072 mole) of 1(+)glutamic acid diethylester hydrochloride and 1.0 milliliter of pyridine in 50 milliliters ofchloroform.

The chloroform solution of l(+)glutamic acid diethyl ester was addedwith stirring to the p- [(2 acetamido-4-hydroxypyrimido[4,5-bl-pyrazine-S-ylmethyl) -acetamidol benzoyl chloride. Ayellow-orange solution immediately formed'with a slight evolution ofheat. The mixture was stirred continuously for four hours and the clearsolution was then allowed to standovernight at room temperature. Thechloroform and some pyridine were removed under reduced pressure leavingabout 4.0 grams of a reddish gum containing N- (p-(2-acetamido-4-hydroxypyrimido [4,5-bl-pyrazine 6 ylmethyl)-acetamidol-ben zoyl) -glutamic acid diethyl ester.

The reddish gum was dissolved in 50 milliliters of 1 N sodium hydroxideand evacuated on the water pump to remove the last of the chloroform.The solution was heated for three hours at a bath temperature of 120 C.and diluted with water to a volume of 100 milliliters. Uponacidification of the solution to pH 2 with concentrated hydrochloricacid, a reddish-orange precipitate separated from solution. Thisprecipitate was collected by centrifugation and was washed successivelywith water containing a small quantity of acetic acid, acetone andether. A yellow-tan product weighing about 1.0 gram and containingapproximately 40% N-(p-[(2-amino-4-hydroxy pyrimido [4,5-bl-pyrazine-B-ylmethyl) -aminol benzoyl) -glutamic acid was obtained asshown by comparison bioassays with natural N-(p-[(2-amino-4-hydroxy-pyrimido [4,5-b] -pyrazine 6- ylmethyl) amino] benzoyl)glutamic acid, a yield of about 36% of theory.

Purification of the crude product by the procedure of Example 1 gave aproduct of about 56% N- (p-[ (2-amino-4-hydroxypyrimido [4,5-b]-pyrazine 6 ylmethyl) -aminol-benzoyl) glutamic acid which had thefollowing characteristic ultraviolet absorption spectrum in pH 10.81solution:

Maxima at 3675 13., E15,?- 206;

2825 Elg 526; 2575 E s 364 A second precipitation from a hydrochloricacid solution followed by crystallization from hot water gave a productcontaining about 70% N- (p-[(2-amino 4 hydrcxypyrimido [4,5-b] -pyrazine6 ylmethyl) amino] benzoyl) -glutamic acid.

Example 4 A mixture of 0.5 gram of p-[(2-acetamido 4- hydroxypyrimido[4,5-b] -pyrazine-6-y1methyl) acetamidol-benzcic acid and 15 millilitersof thionyl chloride was warmed on a steam bath for about five minutesuntil a yellow-orange solution formed. The solvent was removed underreduced pressure, leaving a yellow-orange powder.

The yellow-orange powder was dissolved in 30 milliliters of chloroform,filtered and diluted to 125-milliliter volume with petroleum ether (30-60). The tan precipitate that formed was collected by centrifugation andwas Washed once with petroleum ether. After partial drying, theprecipitation and filtration procedure was repeated to make a total offour times. The very light tan material, still moist with petroleumether, was evacuated in a desiccator over sodium hydroxide. There wassecured 0.41 gram of p- (2acetamido-4hydroxypyrimido [4,5 -bl-pyrazine-fi-ylmethyl) -acetamidol -benzoyl chloride. A ZO-milligramsample was evacuated on the oil pump for micro-analysis.

Analysis-Calculated for C1BH15N6O4C11 C, 52.12; H, 3.65; Cl, 8.55.Found: C, 50.97, 50.76; H, 3.68, 3.80; Cl, 8.76.

To 0.47 gram of p-[(2-acetamido-4-hydroxypyrimido [4,5-b] pyrazine 6ylmethyl) acetamidol-benzoyl chloride precipitated several times fromchloroform solution with petroleum ether Was added a 50-milliliterchloroform solution containing about three equivalents of 1(+)glutamicacid diethyl esterr The clear yellow-orange reaction mixture was stirredfor four hours and then allowed to stand overnight. (The solution ofester was prepared as follows: To 0.86 gram (0.0036 mole) ofl(+)glutamic acid diethyl ester hydrochloride in 30-milliliters of waterwas added 0.3 gram (0.0036 mole) of sodium bicarbonate. This solutionwas extracted with five 25-milliliter portions of chloroform; theextracts were dried over anhydrous magnesium sulfate and then filtered.The chloroform was concentrated under reduced pressure to aSO-milliliter volume.)

The chloroform was removed from the reaction mixture under reducedpressure. The residue was dissolved in 25 milliliters of 1 N sodiumhydroxide and evacuated to remove the last of the chloroform. Thesolution was heated for three hours in an oil bath at (3., cooled anddiluted with water to a 75-milliliter volume. After acidification to pH2-3 with concentrated hydrochloric acid, the precipitate was collectedby centrifugation and was washed with water containing a little aceticacid, then with acetone and finally ether. There was secured 0.42 gramof a yellow-orange product, which was about 70% N (p [(2 amino 4hydroxypyrimido [4,5 bl pyrazine 6 ylmethyl) amino] benzoyD-glutamicacid by comparison bioassay with natural N (p 2 amino 4 hydroxypyrimido[4,5 b] pyrazine 6 ylmethyl) amino] benzoyl) glutamic acid, a yield ofabout 60 of theory.

Of this crude product, 0.39 gram was extracted with two 1.0-milliliterportions of 15% hydrochloric acid. The residue was washed as above anddried. There was secured 0.070 gram of residue. The extracts werediluted to a ZGO-milliliter volume. The precipitate which formed wascollected, washed and dried as above; weight 0.030 gram. To the200-milliliter volume was added sodium acetate until no more precipitateformed. This yellow-orange precipitate was collected by centrifugationand was washed with water containing a little acetic acid, then acetoneand finally ether. This product, weighing 0.23 gram, was essentiallypure N-(p-E (Z-amino-4-hydroxypyrimido [4,5-b] -pyrazine-6-ylmethyl)-aminol benzoyl) -glutamic acid, representing a yield of about 50% oftheory.

Example 5 The preparation starting with 1.0 gram of p- [(2 acetamido 4hydroxypyrimido [4,5-blpyrazine 6 ylmethyl) acetamido] benzoic acid wasrepeated as in Example 3 with the difference that ethylene dichloridecontaining 1.0 milliliter of pyridine was used as the reaction medium.The product weighed 1.04 grams and was approximately 40% N (p [(2 amino4 liydroxypyrimido [4,5-b] -pyrazine-6-ylmethyl)aminol-benzoyl)-glutamic acid by comparison bioassays with natural N (p[(2 amino 4 hydroxypyrimido [4,5-b] -pyrazine-6-ylmethyl)aminol-benzoyl) -glutamic acid, a yield of about 38% of theory.

Purification of the crude product by the method of Example 1 gave aproduct of about 50 to 60% N (p [(2 amino 4 hydroxypyrimido [4,5 b]pyrazine 6 ylmethyl) amino]- benzoylglutamic acid by comparisonbioassays with natural N (p [(2 amino 4 hydroxypyrimido [4,5 bl pyrazine6 ylmethyl)- aminol -henzoyl) -glutamic acid.

Example 6,

Two grams (0.005 mole) of--p-[(2-aoetamido-4- hydroxypyrimido [4,5-b]-pyra zine-6-ylmethy1) acetamidol-henzoic acid in 40 milliliters ofthionyl chloride was warmed on the steam bath until a clear solutionresulted. This solutionwas concentrated on the water pump to leave ayelloworange residue which was evacuated for a short time. To this wasadded dl-methionine ethyl ester in about 100 milliliters of chloroform.The clear reddish-orange solution whichresulted was let stand at roomtemperature for three days.

[The ester solution was prepared as follows: 4.27 grams (0.020 mole) ofdl-methionine ethyl ester hydrochloride was dissolved in 2,0 millilitersof water. To this was added 1.68 grams (0.020- mole) of sodiumbicarbonate in'10 milliliters of water. milliliter portions, ofchloroform. After drying over anhydrous magnesium sulfate, thechloroform solution was concentrated under reduced pressure to about100-mi1liliter volume] The chloroform solution was concentrated underreduced pressure to leave a gummy residue. This was dissolved in 100milliliters of 1 N sodium hydroxide and heated at a bath temperature ofabout 120 for three hours. At the beginning of the heating, thecondenser was removed for a time so any chloroform could escape. Aftercooling, the solution was acidified to pH 2 with concentratedhydrochloric acid. The-.yellow-orange precipitate was collectzd bycentrifugation and was washed five times with water containing a littleacetic acid.

The yellow-orange precipitate was extracted with ill-milliliter portionsof hydrochloric acid and the filtered extracts were precipitated usingfiltered saturated sodium acetate solution. The yellow-orangeprecipitate was collected by centrifugation and was washed with watercontaining a little acetic acid. This procedure was performed threetimes. The final precipitate was Washed with water containing a smallamount of acetic acid until the washes were chloride free and dried invacuo over potassium hydroxide. There was secured 1.6 grams ofyellow-orange N (p [(2 amino-l-hydroxypyrimido [4,5-blpyrazine 6ylmethyl) amino] benzoyl)-dlmethionine, essentially pure, a yield of 72%of theory.

Analysis.Cal-eulated for C19H21O4N7S: C, 51.4.6; H, 4.77; 10,2211.Found: C, 51.73; 1-1, 4.95; N, 21.94.

Example 7 Two grams (0.005 mole) of p-[(2-acetamido-4- hydroxypyrimido[4,5-b] -pyrazine-6-ylmethyl) aeetamidol-benaoic acid was converted tothe chloride as in Example 6. l-Leucine ethyl ester hydrochloride (3.91grams) (0.020 mole) was converted to l-leucine ethyl ester dissolved in100 milliliters of chloroform by the method of Example 6. The chloroformsolution was added to the solid acid chloride. The clear orange-redcolored solution which formed was let stand at room temperatureovernight.

The reaction mixture was worked up as in Example 6. The clearyellow-orange product was purified by the extraction procedure ofExample 6. There was secured 1.43 grams of yelloworange N(p-[2-amino-4-hydroxypyrimido [4,5- bl pyrazine 6-ylmethyl-aminol-benzoyD-lleucine, essentially pure. a yield of 67% of theory.

.lnclysia-Calculatedfor C20H23O4N72 C, 56.46;

This solution was extracted with six 20- i H, 5.45; N, 2 3.05. Found: C,56.42; H, 5.62; N, 23.33.

Example 8 Two grams (0.005 mole) of p-[(2-acetamido-4- hydroxypyrimido[4,5-bl -pyrazine-6-ylmethyl) acetamidol-benzoic acid was converted to.the chloride as in Example 6'. dl-Phenylalanine ethyl esterhydrochloride (4.39 grams) (0.020 mole). was converted todl-phenylalanine ethyl ester dissolved in about milliliters ofchloroform by the method of Example 6. The chloroform .solution Wasaddedto solid acid chloride. Theclear orange-red colored solution whichformedwaslet stand overnight at room temperature.

The reaction mixture was worked up asinExample 6. The crudeyellow-orange product was purified-by the extraction method of Example.6. There was secured 1.35 grams of yellow-orange N (p [(2amino-4-hydroxypyrimido [4,5-blpyrazine 6 ylmethyl) amino] benzoyl)-dlphenylalanine, essentially pure, a yield of 58% of theory.

AnaZysis.-Calculated for Cpl-1210mm C, 60.12; H, 4.61; N, 21.34. Found:C, 60.39; H, 4.85;N, 20.82; N, 20.67

. Example 9 Two grams (0.005 mole) of p -[(2-acetamido-4-hydroxypyrimido [4,5-10] -pyrazine-6.-ylmethyl) acetamidol-benzoic acidwas converted to the chloride'as in Example 6. dl-Valineethyl esterhydrochloride (3.6lgrams) (0.020 mole) was converted to dl-valine. ethylester. dissolved in 100 milliliters of chloroform. by the method. ofEX.- ample 6. The chloroform solution was added to the solidacidchloride. The clear reddish-orange solution which formed was letstand overnight at roomtemperature.

The reaction mixture was worked up as in Example 6. The crudeyellow-orange product was purified by the extraction method of Example6, There was secured 1.41 grams of yellow-orange N -(p [(2-a mi no 4hydroxypyrimido [4,5 0] QYrazine-G-ylmethyl) -aminol -benzoyl) -dl .valine, essentially pure, ayield of 68% of theory.

An alysz's. -Calcu lated for Cl9H2lO4N'lI C, 55.47; H, 5.15; N, 23.83.Found: C, 56.09; H. 5.41; N, 23.5 7.

Example 10 Two grams (0.005 mole) of p-[(2-acetamido- 4-hydroxypyrimido[4,5-bl-pyrazine-6 ylmethyl) acetamido]-benzoic acid was converted tothe chloride as in Example 6. dl-Aspartic. acid diethylester.hydrochloride (4.51 grams) (0.020 mole) was converted to dl-asparticacid diethyl esterdissolved in about 100 milliliters of chloroform. bythe method of Example 6. The. chloroform solution was added tothe solidacid. chloride. The clear reddish solution which formed was let standat25 for eighteen hours.

The reaction mixture was worked up as in Example 6. The crude tan-yellowproduct was purified by the extraction procedure of Example 6. There wassecured 1.24 grams of tan-yellow N-(p [(2 amino- 4 hydroxypyrimido[45-h]- pyrazine-6-ylmethyl) -aminol-benzoyl) d1 as part-i0 acid,essentially pure, a yield of 58% of theory.

Analysz'a-Calculated for CraHwOeNv: C, 50.58; H, 401; N, 22.94. Found:C, 50.40; H, 4.36; N, 23.56.

Example 11 Two grams (0.005 mole) of p-,[(2-acetamido- 4-hydroxypyrimido[4,5-bl-pyrazine-6 lmethchloride as in Example 6. dl-Alanine ethyl esterhydrochloride (3.07 grams) (0.020 mole) was converted to dl-alanineethyl ester dissolved in about 100 milliliters of chloroform by themethod of Example 6. The chloroform solution was added to the solid acidchloride. The clear reddishorange solution which formed was let stand at25 for 48 hours.

The reaction mixture was worked up as in Example 6. The crude yellowproduct was purified by the extraction procedure of Example 6. There wassecured 1.30 grams of yellow N-(p-[(2-amino 4 hydroxypyrimido[4.5-b]-pyrazine-6-ylmethyl) aminl -benzoyl) -dl-alanine, essentiallypure, a yield of 67% of theory.

AnaZysis.Calculated for C17H1'704N72 C, 53.26; H, 4.47; N, 25.58. Found:C, 53.20; H, 4.68; N, 25.41.

Example 12 A mixture of 0.610 grams (0.00152 mole) of p-E(2-acetamido-4-hydroxynyrimido i,5-bl-pyra zine-fi-vmethyl)-acetamidol-benzoic acid and 20 milliliters of thionyl chloridewas heated at reflux for five minutes. The resultin clea solution waseva orated to dryness under reduced pressure yieldingp-[(2-acetamido-4-hyclroxY- pyrimido [45-h] -oyrazine-6-ylmethyl)-acetamidol-benzoyl chloride as a yellow-orange powder.

To the powder was a ded 0 523 gram (0.00165 mole) of lutamylglutamicacid trimethvl ester in 50 mill liters of chloroform. The solution wasallowed to stand for eighteen hours at room temperature. A small amountof dark brown precinitate was then removed by centrifugation: and thesolvent was removed under reduced pressure, leaving a residue containingN-(p-[(2-acetamido--hvdroxvpyrimido [4.5-bl-nyrazine 6 ylmethvl)-acetarnido] -benzoyl) glutamylglutamic acid trimethyl ester.

The residue was washed successively with 100 milliliters of ethyl ether,15 milliliters of 15% hidrochloric acid, and water until the pH of thewash was 6-7. After drying, the residue weighed 0.417 gram.

The residue was then dissolved in 15 milliliters of N-sodium hydroxideand refluxed in a nitrogen atmosphere for one and one-half hours. Thesolution was adjusted to pH 3-4 with dilute hydrochloric acid, and aprecipitate of crude N-(p- [(2-aminol-hydroxypyrimido [4.5-b] -pyrazine-6 ylmethyl) -aminol -benzoyl) -glutamylglutamic acid formed.

The crude precipitate was dissolved in 15% hydrochloric acid and dilutedwith water. Sodium acetate was added to pH 5 until no more precipitateformed. After four such reprecipitations,

washing with water, and drying, the product was assayed for L. casez'activity and S. L. B. activity. The assay value showed 2030 units permilligram for L. 011562 and 330 units per milligram for S. L. R.

Analysis.-Calculated for C24H2SN809I C, 50.52; H, 4.59; N, 19.65. Found:C, 49.28; H, 4.29; N, 18.40.

N (p [(2-amino-4hydroxypyrimido [4,543]- pyrazine 6 ylmethyl)aminol-benzoyl) -glutamylglutamylglutamic acid, assaying 1000 units permilligram for L. casei and 233 units per milligram for S. L. R... couldbe prepared in the same manner, employing glutamylglutamylglutamic acidtetramethyl ester as a starting material. The

ultra-violet absorption spectrum of this product in pH 11 solution showsmaxima at 2550 3., Egg 353; 2800 ii, Egg 326;

Modifications may be made in carrying out the present invention withoutdeparting from the spirit and scope thereof and the invention is to belimited only by the appended claims.

We claim:

1. The process that comprises reacting a compound having the generalformula:

wherein X represents a halogen and R represents an acyl group containingtwo or more carbon atoms, with a completely esterified a-amino-acidester selected from the group consisting of alkyl and aralkyl esters ofthe a-amino acids in the presence of a partially halogenated hydrocarbonselected from the group consisting of chlorinated and brcminated loweralkanes and lower alkenes containing one of the radicals:

wherein X represents the halogen substituent, hydrolyzin the N- (p-(2-acylamino-4-hydroxypyrimido[4,5 b] pyrazme 6-ylmethyl)-acylaminol-benzoyl) -a-amino acid ester thus formed, and recovering anN- (p-[(2-amino-4-hydroxypyrimido [4,5 bl -pyrazine-6-ylmethyl) -amino]benzoy1-aamino acid.

2. The process that comprises reacting a compound having the generalformula:

wherein X represents a halogen and R represents an acyl group containingtwo or more carbon atoms, with a completely esterified e-amino acidester selected from the group consisting of alkyl and aralkyl esters ofthe a-amino acids in the presence of chloroform, hydrolyzing the N-(p-(Z-acylamino-el-hydroxypyrimido [4,5-lol -pyrazine G-ylmethyl)-acylamino] -benzoyl) -a-amino acid ester thus formed, and recovering anN-(p- [(2aminol-hydroxypyrimido [4,540] -pyrazine G-ylmethyl) -aminol-benzoy1) -a-amino acid.

3. The process that comprises reacting p[(2- acetamido--hydroxypyrimido[4,5-bl-pyrazine- G-ylmethyl)-acetamido]-benzoyl chloride with acompletely esteriiied a-amino acid alkyl ester in the presence ofchloroform, hydrolyzing the N- (p [(2 acetamido-4-hydroxypyrimido[4,5-blpyrazine 6 ylmethyl) -acetamido] -benzoyl) -a amino acid alkylester thus formed, and recovering an N- (p-[ (2-amino-4-hydroxy-pyrimido[4,5 b] pyrazine-fi-yli'nethyl) -amino] -benzoy1) a-amino acid.

a. The process that comprises reacting p-[(2- acetamidol-hydroxypyrimido[4,5-bl-pyrazine- S-ylmethyl)-acetamidol-benzoyl chloride with glumaticacid dialkyl ester in the presence of chloroform, hydrolyzing theN-(p[(2-acetami- 13 do 4 hydroxypyrimido [4,5 b] -pyrazine-6-ylmethyl)-acetamido]-benzoyl) -glutamic acid dialkyl ester thus formed, andrecovering N-(p- (2- amino-4-hydroxypyrimido[4,5-b]-pyrazine-6-ylmethyl) -amino] -benzoyl) -g1umatic acid.

5. The process that comprises reacting p-[(2-acetamido-i-hydroxypyrimido [4,5-bl-pyrazine-6-ylmethyl)-acetamido]-benzoyl chloride with glutamylglutamic acidtrialkyl ester in the presence of chloroform, hydrolyzing the N-(p-[(2-acetamido-i-hydroxypyrimido [4,5-bl-pyrazine- 6 ylmethyl) acetamido]-benzoyl) -glutamylglutamic acid trialkyl ester thus formed, andrecovering N-.(p-[(2-amino-4-hydroxypyrimido [4;,5- b] pyrazine 6ylmethyl) amino] benzoyl) glutamylglutamic acid.

6. The process that comprises reacting p-[.(2-acetamido-4-hydroxypyrimido [4,5-bl-pyrazine-6-ylm'ethyl)-acetamido]-benzoyl chloride with glutamylglutamylglutamicacid tetraalkyl ester in the presence of chloroform, hydrolyzing the N-(p- (2-acetamido-4-hydroxypyrimido [4,5-b] pyrazine-6-ylmethyl)-acetamido] -benzoyl) -glut amylglutamylglutamic acid tetraalkyl esterthus formed, and recovering N-(p-[(2-amino-4-hydroxypyrimido [4,5 b]-pyrazine 6 ylmethyl) aminoly-benzoyl) -glutamylglutamylglutamic acid.

7. The process in accordance with claim 6 in which at least two molarequivalents of glutamic REFERENCES CITED The following references are ofrecord in the file of this patent:

UNITED STATES PATENTS Name Date Angler June 8, 1948 OTHER REFERENCESAberholden et al.: Chemical Abstracts, 20, 1994 (1926).

Fieser and Fieser; Organic Chemistry, pp. 235-236, -186, 608, 1944edition, D. C. Heath and Co.

Wolf et al.: J. Am. Chem. Soc, 69, 2753-2759 (1947).

Number

1. THE PROCESS THAT COMPRISES REACTING A COMPOUND HAVING THE GENERALFORMULA:
 9. N - (P- ((2-AMINO-4-HYDROXYPYRIMIDO (4,5B) - PYRAZINE - 6 -YLMETHYL) - AMINO)-BENZOYL)PHENYLALANINE.